J Am Chem Soc. 2016 Oct 12;138(40):13139-13142. doi: 10.1021/jacs.6b07323. Epub 2016 Oct 04.
Journal of the American Chemical Society
Rachel D Cohen, Gary J Pielak
PMID: 27676610 DOI: 10.1021/jacs.6b07323
There are four well-known levels of protein structure: primary (amino acid sequence), secondary (helices, sheets and turns), tertiary (three-dimensional structure) and quaternary (specific protein-protein interactions). The fifth level remains largely undefined because characterization of quinary structure, the transient but essential macromolecular interactions that organize the crowded cellular interior, requires the measurement of equilibrium thermodynamic parameters in living cells. We have overcome this challenge by quantifying the pH-dependence of quinary interactions in living Escherichia coli cells using the B1 domain of protein G (GB1, 6.2 kDa). To accomplish this goal, we buffered the cellular interior and used NMR-detected amide proton exchange to quantify the free energy of unfolding in cells. At neutral pH, the unfolding free energy in cells is comparable to that in buffered solution. As the pH decreases, the increased number of attractive interactions between E. coli proteins and GB1 destabilizes the protein in cells relative to buffer alone. The data show that electrostatic interactions contribute to quinary structure.
